The proposed research plan represents a continuation of our efforts to unravel the complex gene regulation networks responsible for the specification and differentiation of endomesoderm lineages in the ascidian, Ciona intestinalis. Particular efforts will focus on two such lineages: the tail muscles and notochord. The research plan includes three specific aims. First, a combination of bioinformatics methods and experimental assays will be used to identify new enhancers that mediate either notochord-specific or muscle-specific gene expression in electroporated Ciona embryos. Conserved sequence motifs will be identified within coordinately regulated enhancers, and efforts will be made to identify the specific transcription factors that interact with these newly identified motifs. Second, a variety of methods will be used to determine whether the elegant endomesoderm gene regulation network defined in sea urchins is conserved in Ciona. Most of our efforts will focus on the cis- and trans- regulation of two key endomesoderm patterning genes: Krox1 and GataE. Third, newly established GFP visualization methods will be used to characterize the mutant phenotypes produced by overexpressing endomesoderm patterning genes such as Krox1, FoxA, and Brachyury. The successful execution of the proposed research plan will provide the first systematic comparision of a well-defined gene regulation network in two related, but morphologically distinct, animals.